Cell Cycle-Regulated Oscillator Coordinates Core Histone Gene Transcription Through Histone Acetylation

Cell cycle-regulated oscillator coordinates core histone gene transcription through histone acetylation

Christoph F. Kurata,1, Jean-Philippe Lambertb, Julia Petschnigga, Helena Friesena, Tony Pawsonb,c, Adam Rosebrocka, Anne-Claude Gingrasb,c, Jeffrey Fillinghamd, and Brenda Andrewsa,c,2

aThe Donnelly Center, University of Toronto, Toronto, ON, Canada M5S 3E1; bLunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada M5G 1X5; cDepartment of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 3E1; and dDepartment of Chemistry and Biology, Ryerson University, Toronto, ON, Canada M5B 2K3

Edited by Jasper Rine, University of California, Berkeley, CA, and approved August 21, 2014 (received for review July 25, 2014) DNA replication occurs during the synthetic (S) phase of the eukary- Although the roster of histone chaperones and chromatin otic cell cycle and features a dramatic induction of histone gene remodelers involved in histone gene regulation is impressive, the expression for concomitant chromatin assembly. Ectopic production cell cycle oscillator responsible for restricting histone gene acti- of core histones outside of S phase is toxic, underscoring the critical vation to S phase has remained elusive. In this context, we de- importance of regulatory pathways that ensure proper expression cided to revisit the functions of two poorly understood proteins of histone genes. Several regulators of histone gene expression in that physically interact and are involved in histone gene regula- the budding yeast Saccharomyces cerevisiae are known, yet the key tion, suppressor of Ty (Spt)10 and Spt21 (15–21). Spt10 is a DNA- oscillator responsible for restricting gene expression to S phase has binding protein that localizes to the upstream-activation sequences remained elusive. Here, we show that suppressor of Ty (Spt)10, a pu- (UAS) of histone genes (22, 23). Spt10 contains a putative HAT tative histone acetyltransferase, and its binding partner Spt21 are key domain similar to that of general control nonderepressible (Gcn) 5 – determinants of S-phase specific histone gene expression. We show that (19, 24), which was reported to be involved in acetylation of H3K56 Spt21 abundance is restricted to S phase in part by anaphase pro- at histone promoters in vivo (14). However, to date, HAT activity of moting complex Cdc20-homologue 1 (APCCdh1) and that it is re- Spt10 has not been demonstrated in vitro. cruited to histone gene promoters in S phase by Spt10. There, Spt21 is a protein of unknown function that physically inter- Spt21-Spt10 enables the recruitment of a cascade of regulators, acts with Spt10 (19). Like Spt10, Spt21 influences histone gene including histone chaperones and the histone-acetyltransferase transcription as spt21Δ mutants display dramatically reduced general control nonderepressible (Gcn) 5, which we hypothesize lead to histone acetylation and consequent transcription activation. levels of HTA2, HTB2, and HHF2 transcripts in logarithmically growing cells (15, 25). Here we show that Spt21 is a cell cycle oscillator that serves as a master regulator of S-phase–dependent ukaryotic chromosomes are composed of chromatin, which in histone gene expression. We demonstrate that Spt10 is required turn is composed of a fundamental repeated unit of a histone E to establish repression by the recruitment of HIR and HIR- octamer and DNA, the nucleosome. Each histone octamer includes dependent regulators outside of S phase. Furthermore, the ex- two H3-H4 histone dimers flanked on either side by H2A-H2B pression of Spt21 is cell cycle-regulated with levels peaking in dimers. The four replication-dependent (RD) core histones (H2A, S phase, when it is recruited to histone gene promoters by its H2B, H3, and H4) are among the most conserved eukaryotic partner protein Spt10. We use genetic and biochemical experi- proteins, and all characterized eukaryotic genomes carry more than ments to show that the abundance of Spt21 during G1 phase is one gene encoding each core histone protein. Budding yeast contains two copies of each RD histone gene, each arranged in Significance opposite orientation to a gene encoding its partner within the nucleosome: HHT1-HHF1 and HHT2-HHF2,thetwogenepairs DNA replication and histone gene transcription are tightly encoding core histones H3 and H4, and HTA1-HTB1 and HTA2- linked and occur during the S phase of the eukaryotic cell cycle. HTB2, the two gene pairs encoding H2A and H2B (for recent Histone production outside of S phase is highly toxic, under- reviews see refs. 1 and 2). scoring the importance of regulatory pathways that control Histone gene expression is repressed outside of the synthetic histone gene expression. Although various histone regulators (S) phase by a histone chaperone called the histone regulatory (HIR) have been discovered, the molecular mechanisms responsible complex, which is recruited to a poorly defined DNA sequence, the for the spatial and temporal control of histone gene expression negative regulatory (NEG) region, present upstream of three of the have remained elusive. Here, we describe the discovery of four histone-gene pairs in yeast: HTA1-HTB1, HHT1-HHF1,and Spt21 as a long-elusive cell cycle oscillator responsible for HHT2-HHF2 (3–7). HTA2-HTB2, the fourth histone gene pair, does restricting histone gene transcription to the S phase of the not have a NEG region and is regulated in a HIR-independent eukaryotic cell cycle. We show here that Spt21, together with manner. At HIR-dependent promoters, HIR recruits other histone its partner protein Spt10, regulates histone gene transcrip- chaperones, Asf1 and Rtt106, as well as the chromatin boundary tion by enabling the recruitment of a roster of chromatin- protein Yta7 (8, 9) and the remodel structure of chromatin remodeling proteins. (RSC) ATP-dependent chromatin-remodeling complex (10), which together assemble repressive chromatin, blocking recruitment of Author contributions: C.F.K., J.F., and B.A. designed research; C.F.K., J.-P.L., J.P., and H.F. performed research; T.P., A.R., and A.-C.G. contributed new reagents/analytic tools; C.F.K., RNAPII (11, 12). In S phase, this repressive chromatin is overcome, J.-P.L., J.P., H.F., and A.R. analyzed data; and C.F.K., J.F., and B.A. wrote the paper. allowing recruitment of RNAPII and activation of transcription. The authors declare no conflict of interest. After recruitment of RNAPII, the AAA-ATPase Yta7 is important This article is a PNAS Direct Submission. for efficient transcript elongation by evicting histones H3-H4 (11–13). 1Present address: Cancer Research UK London Research Institute, Clare Hall Laboratories, Other chromatin remodelers also have roles in histone gene South Mimms EN6 3LD, United Kingdom. activation, including the SWI/SNF chromatin-remodeling com- 2To whom correspondence should be addressed. Email: [email protected]. plex, which activates NEG-dependent histone genes (14), and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the histone-acetyltransferase (HAT) complex Rtt109-Vps75 (8). 1073/pnas.1414024111/-/DCSupplemental.

14124–14129 | PNAS | September 30, 2014 | vol. 111 | no. 39 www.pnas.org/cgi/doi/10.1073/pnas.1414024111 Downloaded by guest on September 25, 2021 regulated by the anaphase-promoting complex/cyclosome (APC/C) A associated with its activator protein Cdc20-homologue 1 (Cdh1). During S phase, Spt21 accumulates and recruits the Gcn5 HAT to histone gene promoters, where they influence histone gene transcription. Our data reveal an important cell cycle oscillator that links the cell cycle machinery and histone acetylation and explain how the timing of histone acetylation at histone gene B promoters leads to gene activation. C Results Spt10 and Spt21 Recruit HIR and Associated Proteins/Complexes. To explore the mechanism of Spt10-dependent activation of histone gene transcription, we first used affinity purification and mass spectrometry to discover proteins associated with Spt10. Spe- cifically, we used a tandem affinity purification (TAP)-tagged version of Spt10 expressed either at its endogenous locus or from an inducible promoter (GAL1-10) and a modified chromatin immunoprecipitation (mChIP) protocol to identify proteins associated with Spt10 on chromatin (8, 11, 26). We discovered interactions between Spt10 and all HIR subunits (Hir1, Hir2, Hir3, and Hpc2), RSC, as well as most subunits of SWI/SNF (Fig. S1). We did not identify peptides for these Spt10-interacting proteins across several control purifications, and the recovery of peptides for interacting proteins scaled with the amount of Spt10 in our purifications. We performed a similar mChIP analysis with Spt21-TAP and detected SWI/SNF peptides, but only when Spt21 was overexpressed, consistent with its low abundance relative to Spt10 (Fig. S1). We also confirmed the Spt10/Spt21 interaction described previously (Fig. S1)(19). Because we detected all four HIR subunits in our mChIP of Spt10-TAP, we next used chromatin immunoprecipitation (ChIP) to test whether SPT10 is required for recruitment of HIR (Hir1- TAP), the HIR-dependent regulators RSC (Rsc8-TAP), SWI/SNF (Snf6), Rtt106-TAP, and Asf1-TAP, and the transcriptional activator Yta7 (Yta7-TAP) (8, 11, 13) to the promoter region of Fig. 1. The Spt10/Spt21 complex recruits chromatin-modifying proteins and HTA1-HTB1 (Fig. 1A) (11). Consistent with an important function complexes to the HTA1-HTB1 promoter. (A) Schematic representation of the for the Spt10–HIR interaction on chromatin, we observed a strong HIR-dependent HTA1-HTB1 and HIR-independent HTA2-HTB2 histone gene reduction in the recruitment of both negative regulators of histone promoters. UAS sequences (green circles) found in both regulatory regions gene expression—HIR, Rtt106, Asf1, and RSC–as well as activa- (for a comprehensive review see ref. 2). Primer pairs used for ChIP assays are tors of histone transcription–SWI/SNF and Yta7–to the HTA1- shown with black arrows (8, 10, 11). (B) Spt10 and Spt21 recruit histone HTB1 promoter in an SPT10 deletion strain (Fig. 1B). We also regulators to regulatory regions of the HTA1-HTB1 promoter. Hir1-TAP, Rsc8-TAP, Rtt106-TAP, Yta7-TAP, Snf6, and Asf1-TAP strains in the presence saw less dramatic recruitment defects for HIR and associated Δ Δ Δ (wild type) or absence of SPT10 (spt10 )orSPT21 (spt21 ) were cross-linked proteins/complexes in an spt21 mutant (Fig. 1B). This difference using formaldehyde. DNA copurifying with IgG-Sepharose and Snf6 anti- likely reflects the dependence of Spt10 association with histone body/Protein A ChIPs were analyzed for enrichment by qPCR, as indicated in promoters on Spt21 only in S phase (19). We conclude that the the relevant panels. (C) Spt10 recruits Spt21 to the HTA1-HTB1 and HTA2- Spt10–Spt21 complex forms a key scaffold for recruitment of HTB2 regulatory regions. ChIP analyses were performed as described in B important positive and negative regulators of histone gene ex- above. Error bars on the histograms represent SDs from the mean of at least pression to their regulatory regions. three replicate qPCR reactions.

Spt21 Activates Histone Gene Expression During S Phase. To further Δ explore the relationship between Spt21 and Spt10, we used ChIP S phase. Entry into S phase was significantly delayed in an spt21 BIOCHEMISTRY to assay the dependence of Spt21 recruitment on Spt10 at HIR- strain compared with wild-type cells (Fig. 2B), a probable conse- dependent (HTA1-HTB1) and HIR-independent (HTA2-HTB2) quence of the mis-regulation of histone gene transcription. promoters (Fig. 1A). Recruitment of Spt21-TAP to HTA1-HTB1 HTA1 transcription was reduced during S phase (Fig. 2C, and HTA2-HTB2 was abolished in an spt10Δ strain (Fig. 1C, Upper), and HTA2 expression was not activated in an spt21 de- Lower), whereas recruitment of Spt10-TAP was reduced but not letion strain (Fig. 2C, Lower), which supports a function for eliminated in a strain lacking SPT21 (Fig. 1C, Upper), consistent Spt21 in histone gene activation. Deletion of SPT21 also caused with previous results (19). a defect in HTA1 repression during G2 and M phases (Fig. 2C, We next assessed Spt10 and Spt21 protein levels in cells pro- Upper), consistent with a failure to adequately establish the gressing synchronously through the cell cycle. We arrested Spt10- chromatin structure required to repress histone gene transcrip- TAP and Spt21-TAP cultures in G1 phase with α-factor and tion outside of S phase, likely due to defective recruitment released them into fresh medium to resume the cell cycle. Although of Spt10 and HIR (Fig. 1B). Finally, we uncovered both bio- Spt10-TAP levels remained constant through one synchronous cell chemical and genetic evidence of the interdependence of Spt21 cycle (Fig. 2A), Spt21-TAP protein levels were clearly cell cycle levels and H3K56ac (Fig. 2A): (i) accumulation of H3K56ac was regulated, peaking in S phase just before the appearance of acety- delayed and its levels reduced in an spt21Δ strain throughout one lation on lysine 56 on histone H3 (H3K56ac), a chromatin mark cell cycle (Fig. S2A), which may reflect the delay in cell cycle known to be enriched in S phase on newly synthesized histones (Fig. progression seen in an spt21Δ mutant; and (ii) consistent with 2A) (27), consistent with an important activating role for Spt21 in reduced levels of H3K56ac, deletion of SPT21 suppressed the

Kurat et al. PNAS | September 30, 2014 | vol. 111 | no. 39 | 14125 Downloaded by guest on September 25, 2021 A by SPT21 overexpression was exacerbated by deletion of genes that normally repress histone levels through negative regulation of histone transcription (HIR1) or by promoting histone transcript degradation outside of S phase [LSM1, which encodes a component of Lsm1-7–Pat1 complex (29)]. Deletion of HIR1 enhanced the toxicity of Spt21 overexpression, while deletion of LSM1 was lethal in the presence of high Spt21 levels (Fig. 2D). These genetic B C results are consistent with a significant role for Spt21 in regulating histone gene expression, and emphasize the catastrophic consequences of a failure to ensure that high histone protein levels are restricted to S phase. Our results suggest that a peak of Spt21 during S phase is important for appropriate histone gene expression. We next asked whether a canonical KEN box in Spt21 might be involved in its cell cycle-dependent degradation (Fig. 3A). The KEN box is a substrate recognition motif for the anaphase-promoting complex/ Cdh1, a G1-specific E3 ubiquitin ligase composed of 13 distinct core proteins (APC/CCdh1) (30). We performed several experiments to ask if Spt21 is a substrate for APC/CCdh1. First, we saw that Cdh1 influenced Spt21 abundance; elimination of CDH1 caused an increase in Spt21 levels relative to wild type, whereas D overexpression of Cdh1 resulted in reduced levels of Spt21 (Fig. 3 B and C; Fig. S5A). Second, mutation of conserved residues in the KEN box of Spt21 known to be important for KEN-box function caused increased Spt21 levels during G1 phase (spt21-ken-TAP, Fig. 3C). spt21-ken-TAP protein levels were elevated only in G1 phase as revealed using synchronized cells throughout one cell cycle (Fig. S5B), consistent with the function of Cdh1 in degrading Fig. 2. Spt21 levels are cell cycle-regulated and important for histone gene proteins exclusively during late M and G1 phases (30). Third, transcription and S phase entry. (A) Spt21, but not Spt10 protein levels, are we observed a KEN-box–dependent physical interaction between cell cycle-regulated and peak during S phase. Protein levels were monitored Spt21-TAP and HA-Cdh1 (Fig. 3D). To avoid toxic effects of by Western blotting using anti-TAP antibody. Cells were synchronized in G1 overexpressed Cdh1, expression was induced for not more than 30 μ α phase using 5 M -factor and released into fresh medium to resume the cell min. Finally, G1 cells expressing the stabilized KEN-box mutant of cycle. Histone H3 acetylated at lysine 56 (H3K56ac; S and early G2 phase) was analyzed to monitor cell cycle. (B) Spt21 is important for timely S-phase Spt21 (spt21-ken-TAP) had higher levels of transcripts from all entry. A spt21 deletion mutant and an isogenic wild-type control strain were core histone genes (Fig. 3E), and combining the spt21-ken muta- synchronized as in A, and FACS profiles were analyzed throughout the cell tion with deletion of a component of the HIR complex required cycle as indicated. The asterisks mark the entry into S phase. (C) Mis-regu- for repression of histone gene repression was lethal (Fig. 3F). Cell lation of histone gene expression in the absence of SPT21. Cells were syn- cycle experiments revealed that the increased levels of histone chronized as described in A. cDNA was prepared and the ratio of HTA1 or gene transcripts were most prominent in the G1 phase (Fig. S5C). HTA2 transcript to that of ACT1 was determined using qPCR. Error bars in It is not only critical to restrict histone gene expression to the experiments represent SDs from the mean of at least three replicate S phase during an unperturbed cell cycle, it is also important for qPCR reactions. (D) Genetic interactions involving Spt21 overexpression and histone gene expression to be repressed upon genotoxic stress regulators of histone mRNA levels. The indicated strains were spotted in Δ serial 10-fold dilutions onto glucose- or galactose-containing medium (to (31). We saw that both the spt21-ken-TAP mutant and the cdh1 induce SPT21 overexpression) and incubated at 30 °C for 2 d. mutant strain were comparably sensitive to hydroxyurea (HU), which inhibits DNA replication (Fig. 3G). We also observed a clear upregulation of HTA2 transcript levels in a spt21-ken-TAP slow-growth phenotype seen during replication stress or high mutant under HU treatment (Fig. S5D), suggesting that sensi- temperature in strains lacking both putative H3K56ac deacety- tivity to HU might be due to increased histone levels. Also, a lases HST3 and HST4 (28) (Fig. S2B). The elevated H3K56ac double mutant carrying both the spt21-ken-TAP allele and a de- levels in an hst3Δhst4Δ double-mutant strain were restored close letion of YTA7, a key regulator of histone gene expression, which to wild-type levels in an hst3Δhst4Δspt21Δ triple mutant, likely is also important for recovery from genotoxic stress (32), was due to reduced H3 levels (see H3 panel, Fig. S2C). Together, inviable in the presence of HU (Fig. 3G). We conclude that Spt21 protein abundance is regulated and restricted to S phase these results suggest that Spt10 sets up a repressive chromatin Cdh1 structure outside of S phase at the HTA1-HTB1 promoter by by APC/C and that Spt21 degradation is also important to ensure that histone gene expression is not inappropriately acti- recruiting HIR and associated proteins. To activate histone gene vated during genotoxic stress. We note that how Spt21 is pre- transcription, Spt10 recruits and stabilizes its S-phase–specific vented from accumulating during G2 and M phases is currently partner Spt21 at both HTA1-HTB1 and HTA2-HTB2 promoters. unknown. However, because Spt21 does not possess a destruction box (D box), it is unlikely to be a direct target of the G2-M–specific Proper Regulation of Spt21 Is Critical for Normal Cell Cycle Progression. form of the APC, APC/CCdc20. So far, our analysis implicates Spt21 in the coupling of S phase- specific histone gene expression and cell cycle progression. To test Purified Spt21 Is Associated with HAT Activity in Vitro. Previous work this further, we assessed the phenotypic consequences of consti- has shown that Spt10 function in vivo is dependent on its puta- tutive Spt21 expression from the galactose-regulated promoter tive HAT domain and on Spt21 (14, 19), suggesting that Spt21 (GAL1-10). Overexpression of Spt21 caused increased HTA1 and may activate Spt10. So far, evidence for functionality of the HTA2 transcript levels relative to wild type (Fig. S3C) and a sig- Spt10 HAT domain is based on analysis of a mutant derivative of nificant growth defect (Fig. 2D). To explore the relationship be- SPT10, spt10-199, carrying a mutation predicted to create a cat- tween these two phenotypes, we asked if the growth defect caused alytically inactive HAT (Fig. S3A) (19). Consistent with a role for

14126 | www.pnas.org/cgi/doi/10.1073/pnas.1414024111 Kurat et al. Downloaded by guest on September 25, 2021 Spt10-TAP, suggesting that the HAT domain mutation causes A E protein instability, making it difficult to attribute phenotypes specifically to HAT activity (Fig. S3B). We next sought to directly assay HAT activity associated with Spt10 and any dependence on Spt21. To do this, we separately B purified overexpressed Spt10-TAP and Spt21-TAP from yeast (Fig. S6A) and used these proteins in an in vitro HAT assay. Consistent with previous reports (14, 19), we did not detect HAT activity associated with purified Spt10 alone. Surprisingly, we detected a concentration-dependent HAT activity associated with purified Spt21-TAP in vitro against histone H3 and H4 substrates (Fig. S6B). We conclude that Spt21 is associated with a histone H3 and H4 HAT activity that does not appear de- C pendent on Spt10, which is required for its association with F histone gene promoters. In Vitro HAT Activity of Spt21 Is Partly Dependent on Gcn5. As noted earlier, Spt21 does not possess a canonical HAT domain, and we wondered if the Spt21-associated HAT activity that we observed in vitro might be due to a copurifying HAT(s). Although we failed to detect other HATs copurifying with Spt21 in our mass D spectrometry analysis, we used our ChIP assay to assess association of HATs with histone promoters and Spt10. We used tagged versions of Rtt109 (Rtt109-TAP), Hat1 (Hat1-TAP), NuA4 (Esa1-TAP), and Gcn5 (Gcn5-TAP) to assay their association with histone gene promoters. Only Gcn5, a HAT known to be involved in regulation of G1-specific promoters, was clearly G detectable on HTA1-HTB1 and HTA2-HTB2 promoters (Fig. 4A). To determine if association of Gcn5 with histone gene promoters was dependent on Spt21, and because we could not detect Gcn5 associated with Spt21 in our standard mChIP analysis (Dataset S1), we next affinity-purified Gcn5-TAP in a strain in which the spt21-ken mutant was tagged with a HA tag. Fig. 3. APC/CCdh1 targets Spt21 for degradation during G1 phase. (A) Using this strategy, we observed copurification of Spt21 with Schematic representation of Spt21 including the canonical KEN box. (B) Gcn5 (Fig. 4B). Gcn5-TAP also interacts with Spt21-HA wild- Analysis of Spt21 levels in a cdh1Δ strain. Strain bearing Spt21-TAP ± CDH1 type protein, although to a lesser extent than the spt21-ken-HA were grown to log phase, and protein samples were taken at the indicated derivative (Fig. S7A). We also observed that Gcn5 recruitment to time and analyzed by Western blotting. (C) Increased Spt21 protein levels histone promoter chromatin was dependent on Spt21. Elimina- during G1 phase in the absence of CDH1 or in a strain carrying a KEN-box tion of SPT21 had the most dramatic effect on recruitment of mutation. Isogenic wild-type Spt21-TAP and spt21-ken-TAP mutant strains Gcn5 to the HTA2-HTB2 promoter, whereas recruitment to the were arrested with α-factor as described in Fig. 2A. Spt21 protein levels were assayed by Western blotting with anti-TAP antibody. A similar experiment HTA1-HTB1 promoter was only slightly affected (Fig. 4C). This was performed using a Spt21-TAP cdh1Δ strain. In this case, G1-phase cells observation was corroborated by analysis of histone gene ex- were isolated by centrifugal elutriation because the cdh1 mutant strain is pression, which revealed that deletion of GCN5 caused a minor resistant to α-factor (the G1 phase fraction was confirmed by FACS analysis). but reproducible reduction in HTA2 and HTB2 mRNA levels, (D) Spt21 physically interacts with Cdh1 in a KEN-box–dependent manner. Iso- whereas expression of HTA1 and HTB1 were not significantly genic Spt21-TAP, GAL-HA3-Cdh1, Spt21-TAP, GAL-HA3-Cdh1, and spt21-ken-TAP affected (Fig. 4D). Finally, using synchronized cells, we observed GAL-HA3-Cdh1 strains were grown in galactose to induce Cdh1 expression. a defect in activation of HTA2 transcription in gcn5Δ mutants Copurifying proteins were analyzed by Western blotting using anti-HA antibody, during early S phase (Fig. S7B). which detects both the TAP and HA tags. The star marks Spt21-TAP and Because Gcn5 interacts with Spt21 and its deletion impacts the spt21-ken-TAP, and the arrowhead marks HA3-Cdh1. Input samples were incubated with anti-TAP and anti-HA antibodies, respectively. (E) Stabiliza- transcription of HTA2 and HTB2, we wondered if the in vitro HAT activity that we observed for purified Spt21 might reflect its tion of Spt21 in G1 phase causes increased transcription of histone genes. BIOCHEMISTRY Indicated strains were arrested in G1 phase. cDNA was prepared and levels association with Gcn5. Indeed, we failed to detect H3 acetylation of all histone gene pair transcripts were analyzed as described in Fig. 2C (for associated with Spt21 purified from a gcn5Δ mutant, consistent primers see ref. 11). Cells were in G1 phase as determined by budding index. with the known role of Gcn5 as an H3K9 and H3K14 HAT (Fig. Error bars in the experiments represent SDs from the mean of at least three 4E) (33). However, H4 acetylation was still clearly present, which replicate qPCR reactions. (F) Stabilized Spt21 shows genetic interaction with suggests that another H4-specific HAT (or HATs) may be pre- a hir1 deletion mutant. Representative tetrads resulting from sporulation sent at low amounts in the purified Spt21 sample. Consistent of a spt21-KEN-TAP HIR+/ hir1Δ/SPT21+ strain are shown. Spores were in- with this possibility, the observed defect in histone gene tran- cubated for 2 d at 30 °C. (G) Stabilized Spt21 is sensitive to genotoxic stress Δ and interacts genetically with YTA7. Indicated strains were spotted in serial scription in a gcn5 mutant is relatively minor, making it likely 10-fold dilutions onto medium ±0.2 M HU and incubated for 2 d at 30 °C. that Gcn5 operates redundantly with another HAT to regulate Spt21-TAP in a hir1Δ or yta7Δ background behaved like wild type, indicating histone transcription. In an attempt to test this possibility, we no interference of the TAP tag (Fig. S4 A and B). analyzed the stringently purified material used for in vitro assays by mass spectrometry. We failed to detect any copurifying HAT, including Gcn5 or Spt10, in our sample after analyzing biological Spt21 in activating Spt10, the elevated levels of HTA1 and HTA2 replicate purifications (Dataset S2), indicating that interaction mRNA observed when Spt21 was overexpressed were completely with Gcn5, which appears to be biologically meaningful (see suppressed in an spt10-199 strain (Fig. S3C). We note that levels above), and other HATs, may be below the detection limit of of Spt10-199-TAP protein are significantly reduced relative to our method.

Kurat et al. PNAS | September 30, 2014 | vol. 111 | no. 39 | 14127 Downloaded by guest on September 25, 2021 A B we detect associated with Spt21 in vitro. However, associated Gcn5 does not appear to explain the Spt21-associated H4 HAT activity, and we failed to detect other known HATs in our purified Spt21 samples using mass spectrometry. Furthermore, we failed to detect association of the two major H4 HATs, Hat1 or NuA4, with histone promoters (Fig. 4A). However, the relatively mild reduction in HTA2 and HTB2 transcript levels in a gcn5Δ mutant makes it likely that there are redundant HAT(s) involved in histone gene regulation. These unknown HATs might also C D acetylate a nonhistone protein, which might be important in histone gene activation. Although traces of Hat1 or NuA4 not detectable in our mChIP or Western blot-based assays may ac- count for the observed in vitro activity, we cannot exclude the possibility that Spt21 is a previously unidentified H4 HAT that functions specifically on histone promoters during S phase. This idea is highly speculative at this point, and further work will be necessary to identify other Spt21-associated HATs. We propose a model to describe histone gene activation in yeast where Spt10 recruits HIR to histone promoters outside of S phase, establishing repressive chromatin via HIR-dependent EF recruitment of Asf1, Rtt106, RSC, and Yta7. During G1 phase, the APC/CCdh1 prevents Spt21 from accumulating and activating histone transcription prematurely (Fig. 4F). In S phase, when the APC/CCdh1 is inactivated (30), Spt21 accumulates and is recruited to all histone gene promoters by Spt10, where it acetylates H3 and H4, in part by recruitment of Gcn5 and possibly directly or through recruitment of other unidentified HATs. Histone acety- Fig. 4. HAT activity of Spt21 is partly dependent on Gcn5. (A) Gcn5-TAP but lation relieves HIR-dependent repression, permitting SWI/SNF not other HATs associate with the HTA1-HTB2 and HTA2-HTB2 promoters in recruitment and activation of histone gene expression and also vivo. Gcn5-TAP, Rtt109-TAP, Esa1-TAP, Hat1-TAP, and control strains (U.C. activates HTA2-HTB2 via an unknown mechanism (Fig. 4F). means untagged control) were grown to midlog phase, and association with The mechanism for degrading Spt21 during the G2 and M HTA1-HTB1 and HTA2-HTB2 promoters was assessed as described in Fig. 1C. phases remains unknown. However, Spt21 has a functional ho- (B) Spt21 physically interacts with Gcn5. Isogenic GCN5-TAP, spt21-ken-HA,or Cdh1 GCN5-TAP spt21-ken-HA strains were cultured overnight in glucose-containing molog in fission yeast, Ams2, which is degraded by APC/C in

medium YPD and grown to an OD600 nm of 0.8. Gcn5-TAP and copurifying G1 (38) and by SCF (Pof3) in G2 and M phases (39). The spt21-ken-HA was detected by Western blotting using anti-HA antibody, ortholog of Pof3 in budding yeast is the F-box protein Dia2 (40), which detects both the TAP and HA tags. The star marks Gcn5-TAP, and the which may regulate Spt21 in a similar manner. Together, our arrowhead marks spt21-ken-HA. Input samples were incubated using either results also resolve the long-standing question of which DNA- anti-TAP or anti-HA antibodies. (C) Spt21 recruits Gcn5 to the HTA2-HTB2 binding factor recruits HIR and HIR-dependent factors to his- promoter. Strains carrying Gcn5-TAP in the presence (wild type) or absence of SPT21 (spt21Δ) were grown to midlog phase and cross-linked using formal- tone gene promoters; we show that Spt10 is required for HIR dehyde. Association of Gcn5-TAP with the HTA1-HTB1 and HTA2-HTB2 pro- binding to promoters. We note that, although a significant se- moters was examined as described in Fig. 1B.(D)Gcn5influencesHTA2 and quence-based homolog for Spt21 in larger eukaryotes is not HTB2 gene transcription. Isogenic wild-type and gcn5Δ mutants were grown to obvious, the human NPAT protein appears to be a functional midlog phase, and histone transcript levels were assessed as described above homolog. Like Spt21, NPAT activates histone gene transcription. (Fig. 2C). Error bars represent SDs from the mean of at least three replicate NPAT expression peaks in S phase and is important for S-phase qPCR reactions. (E) Gcn5 is responsible for H3 HAT activity associated with entry (41). Interestingly, NPAT bears a putative KEN box in its Δ Spt21. Overexpressed Spt21-TAP was purified from wild-type and gcn5 amino acid sequence, but the biological function of this domain strains, and in vitro assays were performed as described in Fig. S6.(F)Modelfor Spt21 regulation and function as a histone gene regulator. See text for details. has not been explored. Despite these similarities, the importance of multiple regulatory components in histone gene regulation in both yeast and mammalian systems remains poorly understood, Discussion and the conserved elements of this important regulatory pathway Here we show that Spt21 is a key cell cycle oscillator that serves may be most fruitfully dissected using the accessible yeast model. as an important regulator of S-phase–dependent histone gene Materials and Methods expression. We showed that Spt21 is recruited to histone loci by Yeast Strains and Methods. Yeast strains (Dataset S3) and antibodies used in Spt10 where it may be required for acetylation of core histones. this study are described in SI Materials and Methods. Spt10 has not been shown previously to have HAT activity, although it contains a putative HAT domain. We purified both mChIP Affinity Purification for Mass Spectrometry and in Vitro HAT Assays. Spt10 and Spt21 and detected a copurifying HAT activity specific One-step mChIP affinity purification coupled to mass spectrometry analysis for histone H3 and H4 substrates with Spt21 but not with Spt10. was performed as previously described (26) with minor modifications (SI The in vitro HAT activity in our Spt21 preparations was sur- Materials and Methods). prising, given that an identifiable HAT domain is not present in Spt21. The identification of noncanonical HATs is not un- ChIP and Quantitative PCR Analyses of Histone Gene Transcription. ChIP, cDNA precedented. Rtt109, a known H3K56 HAT (34–36), also has no synthesis, and quantitative PCR (qPCR) to analyze histone mRNA levels were conducted as described (11). typical HAT domain detectable by primary amino acid sequence inspection; however, structural analysis revealed that Rtt109 is TAP Pull-Down Assay. Two hundred milliliters of logarithmically growing cells related to the CBP/p300 family of HATs from mammalian cells (OD at 600 nm ∼1) were harvested, frozen in liquid nitrogen, and processed (37). We found that Gcn5 accounts for the H3 HAT activity that as described in SI Materials and Methods.

14128 | www.pnas.org/cgi/doi/10.1073/pnas.1414024111 Kurat et al. Downloaded by guest on September 25, 2021 In Vitro HAT Assays. In vitro HAT assays were performed using core histones laboratory space for in vitro assays, and Jason Lieb for critically reading the (Millipore) and C14-labeled acetyl-CoA (60 mCi/mmol) and purified Spt10, Spt21, manuscript. This work was supported by Grant MT-11206 (to B.A.) from the or both. Proteins were separated by SDS/PAGE and stained with GelCode Blue Canadian Institutes of Health Research (CIHR). C.F.K. was supported by staining reagent before preparation for autoradiography. Gels were dried and a European Molecular Biology Organization long-term fellowship; J.P. by an Austrian Schroedinger postdoctoral fellowship; and J.-P.L. by a CIHR exposed to a phosphoscreen for 4 d. Radioactivity was detected using a Typhoon Research postdoctoral fellowship. Work in the A.-C.G. and T.P. laboratories scanner. Gels were dried and exposed to a phosphoscreen for 4 d. was supported by CIHR Operating Grant MOP 123322. Work in the J.F. laboratory is supported by a Natural Sciences and Engineering Research ACKNOWLEDGMENTS. We thank C. D. Allis for providing anti-H3K56ac Council Discovery Grant. B.A. is a Fellow of the Canadian Institute for antibody, F. Sicheri for tobacco etch virus protease, J. Greenblatt for providing Advanced Research.

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